Abstract 4862: Whole genome amplification allows for testing of valuable specimens by array comparative genomic hybridization

Abstract

Abstract Many cancer clinical specimens are obtained in minute quantities and in varying states of degradation; for example, when they are formalin or ethanol fixed. In addition, many investigators seek tests on multiple genomic analytes and frequently insufficient DNA remains for all inquiries after the initial priority testing. To accommodate all clinical scientific inquiries, the Sigma GenomePlex whole genome amplification (WGA) protocol has been evaluated as a means to produce sufficient genomic material for array comparative genomic hybridization (aCGH) downstream testing. However previous studies, including our own, have shown that testing degraded samples with WGA coupled to oligo CGH arrays resulted in decreased data quality. We contend this could be due to unequal genomic representation in the WGA product. Our objective was to assess the importance and efficacy of using correctly matched reference DNA and to compensate for the WGA amplification bias, potentially improving aCGH results. We obtained DNA from ethanol-fixed prostate tissues in which both tumor and normal tissue compartments were separately isolated by laser capture microdissection or by directed needle-core sampling from formalin-fixed paraffin embedded blocks. We sought to optimize the Sigma WGA protocol by testing the following parameters: source of reference DNA (commercial versus matched normal), WGA cycle number, and the use of size-exclusion columns post-WGA. Quality control measures for the purified WGA product included electrophoresis (gel and Agilent Bioanalyzer), and a multiplex PCR to determine the extent of DNA genomic representation across the WGA product. ChromaSpin columns were used to reduce the number of smaller WGA amplicons prior to aCGH. WGA products were assayed with Agilent 8×60K CGH microarrays and chromosomal aberrations and data quality were assessed using BioDiscovery Nexus software. Data will be presented comparing the effectiveness of using sheared reference DNA as opposed to matched normal DNA, the use of QC methods prior to hybridization, any benefit in removing small WGA DNA products, and varying the WGA cycle number. We continue to evaluate how these modifications can bring improvement to WGA when coupled to aCGH, because there is promise that this method could expand the genomic testing opportunities for valuable and rare clinical specimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4862. doi:10.1158/1538-7445.AM2011-4862